EP1089754A2

EP1089754A2 - An antimicrobial effective against gram-positive pathogens

Info

Publication number: EP1089754A2
Application number: EP99928186A
Authority: EP
Inventors: Reynolds Paul Ross; Colin Hill
Original assignee: University College Cork; Teagasc Agriculture and Food Development Authority
Current assignee: University College Cork; Teagasc Agriculture and Food Development Authority
Priority date: 1998-06-22
Filing date: 1999-06-22
Publication date: 2001-04-11
Also published as: AU4529699A; WO1999066949A2; WO1999066949A3

Abstract

An antimicrobial which is effective against a range of Gram positive organisms is described. The antimicrobial is the bacteriocin lacticin 3147, which is shown to be effective both in vivo and in vitro against a range of Gram positive organisms.

Description

An Antimicrobial Effective Against Gram-Positive Pathogens

The present invention relates to an antimicrobial agent which is effective against gram-positive human pathogens. In particular, the invention provides the use of the bacteriocin lacticin 3147 in the preparation of a medicament for the treatment of infections caused by Gram +ve organisms, and to pharmaceutical compositions containing lacticin 3147.

Prior art

The emergence of antibiotic resistant pathogens has long been recognised as a significant challenge to those engaged in clinical research, and intensive research efforts have been devoted to developing alternative antimicrobial agents. One possible avenue lies in the exploitation of the class of natural antimicrobials termed bacteriocins. Bacteriocins are antimicrobial peptides which can have a broad spectrum of inhibition. Some undergo substantial post-translational modifications, e.g. nisin which is a 34 amino acid peptide containing a number of unusual amino acids, including dehydrated residues and five cross-linking lanthionine residues (Jack et al., 1995). This bacteriocin belongs to a class of bacteriocins termed lantibiotics, to reflect both the presence of these unusual residues and its broad host range antimicrobial activity.

A recently identified bacteriocin, which differs from nisin in that it is composed of two components, both of which are essential for its activity, is lacticin 3147. Lacticin 3147 is produced by the GRAS (generally regarded as safe) organism Lactococcus lactis DPC3147 (Ryan et al., 1996). The genetic determinants which encode lacticin 3147 are encoded on a 60.2 kb plasmid, pMRCOl . Lacticin 3147 is the subject of PCT Patent Application No. PCT/IE96/00022, published as WO 96/32482. Lacticin 3147 exhibits a wide host range, though confined to Gram positive organisms, including foodborne pathogens such as Clostridium botulinum, Listeria monocytogenes, Bacillus subtilis and Bacillus cereus. An important distinction between nisin and lacticin 3147 is that the latter is effective at physiological pH. In contrast, nisin displays poor solubility (and thus poor activity) at pH 7, which has to date limited its clinical applications in the treatment of human disease.

The mode of action of lacticin 3147 has been recently determined in studies involving sensitive strains of B. subtilis, L. monocytogenes, and L. lactis (McAuliffe et al, 1998). In brief, lacticin 3147 is a membrane-active pore-forming complex, which exhibits a bactericidal mode of action, causing cell death but not cell lysis. Energised cells are more sensitive, suggesting that the presence of a proton motive force (PMF) promotes the interaction of the bacteriocin with the cytoplasmic membrane, leading to the formation of pores. The pores are selective for ions, in particular potassium and inorganic phosphate. The rapid efflux of ions from the cell results in the immediate dissipation of the membrane potential, and hydrolysis of cytoplasmic ATP as the cell attempts to reaccumulate these ions. This eventually leads to collapse of the pH gradient, resulting ultimately in cell death.

The investigation of an application for lacticin 3147 in the prevention of mastitis in dry cows has produced very encouraging results (Ryan et al, 1998). The bacteriocin was found to be bactericidal against a range of mastitis-causing streptococci and staphylococci, and, when incorporated into a teat seal product, showed considerable potential for the prevention of mastitis.

Previous studies of lacticin 3147 concentrated on its use in fermented foods. In this application, the anti-bacterial activity was demonstrated against drug-resistant pathogens in a buffer system at pH7. This neutral pH is a much better approximation of the human body than previous test systems e.g. foodstuffs. The present invention thus provides a number of applications which are not obvious in the light of previous studies where activity at neutral pH was not demonstrated.

Furthermore, the multiplely drug-resistant organisms such as MRSAs (methicillin-resistant Staphlococcus aureus) are extremely hardy, resistant to a number of antibacterial agents. It could thus not be predicted that lacticin 3147 would be effective against these organisms.

Studies on the effect of lacticin 3147 on mastitis could not be used to predict the effectiveness of this agent on other organisms, as disclosed herein. Lacticin 3147 was isolated from an organism found in milk and mastitis affects the milk-producing gland. One might expect a milk-borne agent to effect an organism found in milk or related to milk production. One could not predict with certainty that the agent would be an effective against non-milk-related organisms.

In this study, lacticin 3147 was investigated for its ability to inhibit a number of Gram positive pathogens, including two methicillin-resistant S. aureus (MRSA) isolates, vancomycin-resistant Enterococcus (VRE), penicillin-resistant Pneumococcus (PRP), Propionibacterium acne and Streptococcus mutatis. The pathogens were chosen to represent organisms with differing sites of infection, including respiratory, meningial, skin, oral, wound and cardiac; as well as including some of the more problematic antibiotic-resistant strains.

Object of the Invention

It is thus an object of the invention to provide an antimicrobial agent effective against Gram-positive bacteria and in particular effective against multiplely drug- resistant organisms such as MRSAs.

Summary of the Invention

According to one aspect of the present invention there is provided use of lacticin 3147 in the manufacture of a medicament for the treatment or prevention of infections caused by Gram-positive bacteria. Suitably the bacteria are multiplely drug-resistant organisms and in particular antibody-resistant bacteria. Suitably the Gram-positive bacteria are selected from Enterococcus, Staphylococcus aureus, Pneumococcus, Propionibacterium acne, Streptococcus mutans, Listeria monocytogenes, Clostridium perfringens, and Cloistridium dificile. The bacteria may be human pathogens.

In a further aspect the invention provides a pharmaceutical composition for the treatment or prevention of infections caused by Gram-positive bacteria comprising lacticin 3147. Pharmaceutical compositions may further comprise a lacticin 3147 induced-bacteriocidal-enhancing amount of glucose.

The pharmaceutical composition may be adapted for topical, parenteral, oral, sub-cutaneous or intra-venous application. The composition may be a selected from mouth wash, a toothpaste, a topical skin preparation including those used for acne treatment, an antiseptic soap, an inhaler, an intra-venous application, an oral ingestion preparation or an antiseptic wipe or the like.

The pharmaceutical composition may further comprise a lacticin 3147 induced- bacteriocidal-enhancing amount of glucose.

In a still further embodiment the invention provides a method of treatment of the human or animal body by the application of lacticin 3147 for the treatment or prevention of bacterial infection, particularly infection by Gram +ve organisms. The method of treatment may also comprise use of a lacticin 3147 induced-bacteriocidal-enhancing amount of glucose.

Figure Legends

The invention will be described further in relation to the accompanying drawings which:

Figure 1. Inhibitory action of lacticin 3147 against the gram-positive species L. lactis subsp. cremoris HP (A), MRSA 13 (B), MRSA 148 (C), penicillin-resistant Pneumococcus (D), vancomycin-resistant Enterococcus (E), P. acne (F), and 5. mutans (G) illustrating the difference in sensitivity of the test strains.

Figure 2. Bactericidal effect of lacticin 3147 on the viability of: (A) MRSA 13, (B) MRSA 148, (C) penicillin resistant Pneumococcus, (D) vancomycin-resistant Enterococcus, (E) Propionibacterium acne, and (F) Streptococcus mutans. (■) no addition, (♦) addition of lacticin 3147 at a concentration of 20,000 AU/ml. Data points along the horizontal axis represent 0% survival. Data points represent the average of experiments performed in duplicate.

Figure 3. Bactericidal effect of lacticin 3147 on the viability of: S. mutans (A), and MRSA 148 (B). (■) no addition, (♦) addition of lacticin 3147 at 20,000 AU/ml. Data points represent the average of experiments performed in duplicate.

Figure 4. Recovery of deliberately infected 5 aureus 5246 infused at a dose of 1,500- 1,800 cfu to quarters of lactating cows containing either teat seal only or a teat seal/lacticin 3147 formulation.

Figure 5. Recovery of deliberately infected S. aureus DPC5246 infused at a dose of 6,800 cfu to quarters of lactating cows containing either teat seal only or a teat seal/lacticin 3147 formulation.

Figure 6. Daily incidence rate of new clinical infections in sealed quarters (□) and quarters treated with seal plus lacticin (■).

Example

Materials and methods

Bacterial strains and culture conditions: The bacteriocin producer L. lactis subsp. lactis DPC3147 was grown at 30°C in M17 (Oxoid Ltd., Basingstoke, Hampshire, England) supplemented with 0.5% (w/v) glucose, as was the standard indicator strain L. lactis subsp. cremoris HP. Pathogenic strains included: vancomycin resistant Enterococcus (VRE) (Beaumont Hospital, Dublin, Ireland), grown in Tryptone Soy Broth (TSB) (Difco Laboratories, Detroit, USA) supplemented with 0.6% (wt/vol) yeast extract (Oxoid); Methicillin resistant Staphylococcus aureus (MRSA) 13 ε 148 (Mercy Hospital, Cork, Ireland), grown in Brain Heart Infusion (BHI) broth (Oxoid); Penicillin-resistant Pneumococcus (PRP) 856 (Mercy Hospital), grown in BHI broth; Propionibacterium acne (ATCC6919 American Type Culture Collection, Maryland, USA), grown in Reinforced Clostridial broth (RCB) (Oxoid); and Streptococcus mutans 257 (Professor W. Bowen, University of Rochester, New York, U.S.A.) grown in TSB. All pathogens were grown at 37°C without aeration.

Preparation of Lacticin 3147:

The concentrated preparation of lacticin 3147 used for these studies was prepared as follows; TY broth (β-glycerophosphate at 19 g/L, glucose at 10 g/L, yeast extract at 5 g/L, tryptone at 2.5 g/L, MgSO₄7H₂0) at 0.25 g/L, MnSO₄4H₂O at 0.05 g/L, pH 6.75) was cleared of contaminating proteins which bind to hydrophobic binding XAD-16 beads (Sigma) by passing it through 50 g of beads. Lactococcus lactis

DPC3147 was then propagated in this TY broth overnight at 30°C. The cells were removed by centrifugation at 12,000 rpm. The bacteriocin-containing supernatant was incubated with 25 g XAD-16 beads with agitation for 30 min, at which point a further 25g XAD-16 beads were added, and incubation with agitation was allowed to proceed for a further 30 min, allowing the bacteriocin to bind. The beads were then washed with distilled water, followed by washes with 40% ethanol, and the bacteriocin was subsequently eluted with 70% isopropanol, lOmM acetic acid, pH 2. The isopropanol was removed by rotary evaporation, and the resulting bacteriocin-containing liquid freeze-dried. Finally the bacteriocin was resuspended in 2.5 mM sodium phosphate buffer pH 7, and its activity determined using the agar well diffusion assay. Determination of Bacteriocin Activity:

Bacteriocin activity was determined by the agar well diffusion assay, as described by Ryan et al, 1996. Molten agar at 48°C was seeded with the indicator strain L. lactis subsp. cremoris HP (50 ml of an overnight culture per 20 ml agar), dispensed into sterile petri-dishes, and allowed to solidify. Wells of approximately 4.6 mm in diameter were made, and 50 ml aliquots of a two-fold serial dilution of the bacteriocin preparation were dispensed into the wells. After overnight incubation at 30°C, bacteriocin activity was calculated as the inverse of the last dilution that gave a definite zone of clearance after overnight incubation. Activity units were expressed per millilitre (1/dilution x 20).

Bactericidal effect of Lacticin 3147 on Pathogens:

The bactericidal effect of lacticin 3147 on the six pathogens was investigated by two methods. (A) Initially agar well diffusion assays were performed. These were carried out as described above, except that the agar was seeded with the pathogenic strain. (B) Time-kill curve studies were then performed on the pathogens. Sensitive cells were inoculated at 10% (from an overnight culture) and grown to mid-exponential phase. The cells were washed, resuspended and diluted in 2.5 mM sodium phosphate buffer, pH 7, supplemented with 10 mM glucose, so that upon addition of bacteriocin at 20,000 AU/ml the bacterial count was 10 to 10⁶ cfu. An equal number of cells in buffer without bacteriocin was used as a control. Samples were then taken at appropriate intervals over a 2 hour period to determine the viable cell count.

Formulation of Teat Seals containing Lacticin 3147

An internal intramammary teat sealer similar in composition to a commercial teat seal product currently on the market (Teat seal™; Cross Vetpharm Group, Ltd., Dublin) was used for this study. (A teat seal comprises a heavy inorganic salt in a paraffin/wax base which forms a plug in the teat sinus and acts as a physical barrier to infection). Teat seals containing lacticin 3147 were prepared as described by Ryan et al (1998) except the teat seal was blended with 1% (wt/wt) Tween 80 and left overnight prior to the addition of a lacticin 3147 concentrate (lOOμl/gram of teat seal/Tween80). The blended formulation was filled into a sterile 4 ml syringe and stored at 4°C until use.

Preparation of Bacterial Challenge Cultures

S. aureus DPC5246 was used as the challenge organism to test the efficacy of lacticin 3147 in vivo. One bead of the stock culture was removed and streaked over the surface of an ABA plate and incubated overnight at 37°C. Individual colonies were subcultured from the plate into 10 ml of BHI broth and incubated for 6 h at 37°C. The number of viable cells was counted and then diluted to the required number of colony forming units (cfu) per ml in 10% sterile antibiotic-free skim milk. The diluted culture was stored in 10 ml aliquots at -20°C until required.

In vivo effect of lacticin 3147 formulation against 5. aureus

A novel method to assess the effect of teat seal plus lacticin 3147 formulation on the survival of 5. aureus in the teats of lactating cows was investigated. Animals were chosen for the artificial challenge experiments using udder health status as the criterion for selection. Cows were selected with udders free of pathogens in two foremilk samples collected five to eight days apart with no clinical signs of mastitis in either the udder or in the foremilk (clots, abnormalities, etc.) and somatic cell counts (SCC) less than 120,000/ml (using current and historical SSC data). Udder quarters of lactating cows were randomised into control (untreated) and treatment (teat seal plus lacticin 3147) quarters. Prior to infusion of the teat seals, the tip of each teat was disinfected with a cotton wool swab soaked in methylated spirits. After morning milking, 2 teats in each cow were infused with teat seal plus lacticin 3147 formulation (4g) and the 2 remaining teats in each cow were used as untreated controls. The teat seal was not manipulated in the teat after infusion, so as to allow it to form a plug in the teat sinus and duct. Two hours later all the teats were inoculated to a depth of 17 mm with the S. aureus DPC5246 challenge inoculum using a syringe with a blunted smoothed tip to prevent injury to the teat. Cows were not milked again until the next morning (approximately 18 h later). Teat seals were then removed from each udder quarter, foremilk samples were taken in an aseptic manner from all challenged quarters and the microbiological status assessed.

Milk or dry-period secretions from individual udder quarters were microbiologically assessed by streaking out a loopful (approx. 10 μl) on separate quandrants on the surface of ABA plates and incubating aerobically for 16 h at 37°C.

In vivo effect of Lacticin 3147 on Strep, dysgalactiae

Sixty-eight uninfected udder quarters were selected from 18 cows. Udder quarters were defined as uninfected at drying off if (1) they were free of pathogens in each of two foremilk samples collected 5 to 8 days apart and (2) there were no clinical signs of mastitis in either the udder or the foremilk. Cows were also selected free of teat skin lesions. After the last milking of the lacticin, 33 teats were infused with seal, and 35 were infused with seal plus lacticin 3147. Four teats were not infused due to an insufficient supply of sealing material. Within-cow treatment comparisons were made using treatment pairs selected at random of either the right front and right hind or left front and left hind teats. Three days after infusion, the 68 treated teats were inoculated with Strep, dysgalactiae M using approximately 1.5 x 10 cfu per teat.

Streptococcus dysgalactiae M was selected as the challenge organism because previous in vitro studies showed that lacticin 3147 was effective against this pathogen. This isolate, classified as Strep, dysgalactiae spp. dysgalactiae by SDS-PAGE total protein profiling (BCCM™ Culture Collection; Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium) was previously recovered from a case of clinical mastitis and had been preserved in a microbiological bead storage system (Protect; bacterial preservers; Technical Service Consultants Ltd., Lancashire, England) prior to the start of this study.

At the beginning of the experiment, one bead was removed from a storage vial, streaked on the surface of an aesculin blood agar plate (ABA), and incubated for 16 h at 37°C, The ABA was prepared from blood agar base #2 (Lab M, Bury, England) to which 0.1% aesculin and 7% citrated whole calf blood were added. Two hundred fifty millilitres of brain heart infusion broth (Oxoid Ltd., Hampshire, England) were inoculated with the Strep, dysgalactiae M culture and incubated at 37°C for 8 h. A total bacterial count was carried out on the 8-h stock, which was then diluted to produce a working concentration of 1.5 x 10 cfu/ml in 10% sterile antibiotic-free skim milk. A 0.1-ml aliquot of skim milk containing 1.5 x 10 cfu of streptococci was inoculated into treated and control udder quarters via the teat canal and deposited into the teat sinus at a depth of 17 mm from the tip of the teat. This experimental technique was used primarily to demonstrate the combined effect of the lacticin 3147 with the protection afforded by the teat seal alone.

Samples of secretions (0.1 ml) collected from clinically affected udder quarters were streaked on the surface of ABA plates and incubated aerobically at 37°C fir 24 h. The milk or dry period secretion samples collected from the four quarters of each cow (at the beginning and at the end of the experiment) were streaked on separate quadrants of a single ABA plate.

Following inoculation, the cows were observed twice daily (morning and evening) for signs of clinical mastitis (swelling, hardness, heat, and pain). These observations were conducted in a blind fashion with no knowledge of test and control quarters. The seals were removed from clinically affected quarters by hand-stripping, and samples of secretions, containing seal material were collected aseptically for microbiological analysis. Secretions containing clotted material and shedding mastatic pathogens were classified as clinical. These quarters were treated with intramammary antibiotics (indicated for use in lactating cows) until the clinical signs disappeared and quarters returned to normal. On the last day of the experiment, (8 d after inoculation), samples of secretion, which also contained recovered teat seals were collected both for microbiological analysis and also to measure the persistence of the seal in the remaining uninfected quarters. All inoculated teats were infused with long-acting (dry cow) intramammary antibiotics at the end of the trial to eliminate possible residual contamination associated with the challenge strain. The presence of Strep, dysgalactiae in nonclinical samples of secretion collected at the end of the experiment were recorded as bacteria surviving in the teats as a result of the bacterial challenge. Representative isolates were compared with the challenge Strep, dysgalactiae M strain using randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR) analysis.

Results

In vitro effect of lacticin 3147 on pathogens

Initial trials indicated that lacticin 3147 displays limited activity against the target bacteria on agar well diffusion assays, in comparison to a standard indicator strain, !, lactis subsp. cremoris HP (Figure 1). However, when time-kill curve studies were performed in 2.5 mM buffer using washed, actively growing cell suspensions a more efficient inhibitory effect was observed. Since previous studies using concentrated lacticin 3147 have demonstrated that the presence of glucose at a concentration of 10 mM dramatically increases the killing efficiency of the bacteriocin (McAuliffe et al, 1998), all experiments were performed in the presence of 10 mM glucose at pH 7. Initial studies were performed using 1,200 AU/ml lacticin 3147. This level of activity proved to be ineffective against any of the pathogenic strains. However, increasing the concentration to 20,000 AU/ml gave more dramatic results. Both MRSA strains exhibited a gradual decrease in numbers over time when exposed to this level of bacteriocin, resulting in a 4.5 log kill within 2 hours (Fig.2 (A) and (B)). A more rapid killing effect was observed when two other antibiotic-resistant strains, a vancomycin- resistant Enterococcus, penicillin-resistant Pneumococcus, and P. acne were exposed to 20,000 AU/ml bacteriocin. Within 30 minutes a complete kill was observed (Fig. 2 (C), (D) and (E)). Greater than 10 cells were killed within 90 minutes when S. mutans was exposed to 20,000 AU/ml lacticin 3147 (Fig. 2 (F)).

When much higher initial cell numbers were employed in these assays, a less dramatic kill was observed (Fig. 3.). While the log-reduction was less impressive, the total number of organisms killed was higher in these experiments. This almost certainly reflects the mode of action of the bacteriocin. Those cells receiving a lethal dose will probably retain the bacteriocin in the cell membrane, thus rendering that bacteriocin unavailable to kill further cells. The surviving cells do not represent resistant isolates and are sensitive to further applications of the bacteriocin preparation. Indeed, we have never observed spontaneous resistance to lacticin 3147 among any of the sensitive strains tested.

Effect of a Teat Seal Plus Lacticin 3147 Formulation on the Survival of 5. aureus in Lactating Cows

Animal Trial 1 :

In trial 1, 10 lactating animals were selected and 50% of the quarters were infused with a 4g fill of the teat seal plus lacticin 3147 formulation containing 16,384 AU of bacteriocin, and the remaining 50% were untreated. An inoculum of 0.1 ml containing approximately 1,500 cfu of viable S. aureus DPC5246 cells was introduced into each teat and left for 18h. Viable S. aureus were recovered from all (100%) of both the untreated udder quarters and quarters containing the teat seal/lacticin 3147 formulation (Table 1). These data indicated that a teat seal/lacticin 3147 formulation containing 16,384 AU per tube did not permit a significant reduction in the incidence of teats shedding the challenge organism relative to control teats within the exposure period of the trial.

Animal Trial 2: In a further trial, 16 cows were selected. Based on SSC data and microbiological analyses, 59 quarters were deemed suitable for the trial. From these, 29 quarters were infused with the lacticin 3147 plus teat seal formulation containing 32,768 AU of bacteriocin, one was infused with teat seal containing only 1% Tween 80 and the remaining 29 quarters served as untreated controls. All 59 quarters were infected with 0.1 ml of S. aureus DPC5246 using an inoculum level of 1,500-1,800 cfu/0.1 ml. The S. aureus challenge survived in 18 of the 29 control quarters (62.1%; Table 1). In contrast, the S. aureus challenge was recovered from only 4 of the 29 teats (14% recovery) in quarters containing the teat seal plus lacticin 3147 formulation. The S. aureus challenge survived in the quarter containing the mixture of teat seal and 1% Tween 80. In addition, S. aureus isolates in the foremilk samples were enumerated to assess if a difference in the recoveries between treatments had occurred. With the exception of one quarter, the number of viable S. aureus recovered from the treated quarters was noticeably reduced relative to the untreated quarters (Fig. 4). Overall the presence of the teat seal plus lacticin 3147 formulation significantly reduced (P<0.001) the recoveries of S. aureus. These data indicate, that in addition to reducing the number of teats shedding viable S. aureus, the teat seal plus lacticin 3147 formulation also reduced the numbers of challenge organisms in those teats where S. aureus cells survived.

Animal Trial 3:

A further study using 10 lactating cows was undertaken. Of the 40 udder quarters used for this study, 20 were used as the control group (untreated) and the remaining 20 (treatment group) were infused with the teat seal plus lacticin 3147 formulation containing 32,768 AU per tube. For this trial a higher inoculum level of 6,800 cfu/0.1 ml was used. The S. aureus challenge was recovered from 17 out of 20 of the untreated udder quarters (85%), whereas 11 out of the 20 (55%) teats infused with the teat seal plus lacticin 3147 formulation were still shedding the challenge organism after about 18h (Table 1). As in trial 2 and again with the exception of one teat, significantly lower recoveries (P<) of the challenge organism were observed for teats which had contained the bacteriocin preparation relative to the untreated controls (Fig.

5).

In vivo effect of Lacticin 3147 on Strep, dysgalactiae

In all, 16 clinical cases of mastitis developed in the sealed quarters as compared with 3 in the quarters infused with seal plus lacticin 3147 (Table 2). Subsequently, Strep, dysgalactiae was isolated from 17 of these infected quarters (14 in sealed teats and 3 in the teats treated with seal plus lacticin 3147). Staphylococcus aureus was isolated from the 2 remaining infected quarters treated with the teat seal along. When all remaining nonclinical quarters were sampled at the end of the experiment, Strep. dysgalactiae was recovered from 6 quarters that had been treated with seal alone but not from the quarters treated with seal and lacticin 3147 (Table 2). The daily incidence of new clinical infection caused by the challenge strain is presented for each treatment group in Figure 6. Of the 14 teats containing seal only that became infected with the challenge strain, 12 (86%) had developed clinical mastitis by d-2 following the bacterial challenge. The first clinical case of mastitis in the quarters treated with seal plus lacticin 3147, however, was detected 4 d after the introduction of the challenge.

RAPD PCR profiles for 14 of the Strep, dysgalactiae isolates recovered from the quarters treated with seal and 2 of the Strep, dysgalactiae isolates recovered from quarters treated with seal plus lacticin 3147 confirmed that the infections were caused by the challenge strain. One of the 3 Strep, dysgalactiae isolates recovered from the quarters treated with seal plus lacticin 3147, however, was different from the challenge strain. Combining the information obtained from these analyses, it was shown that there were 14 clinical cases of mastitis caused by Strep, dysgalactiae M in sealed quarters, and only 2 cases in the quarters treated with seal containing lacticin 3147. The difference in new infection caused by the challenge strain between the treatments was significant (P<0.001). When the Cochran-matched pairs teat was applied to these data there was also a significant difference between treatments (p<0.001). Time survival distribution analysis between the rate of occurrence of each clinical event again showed that the treatments were different (p<0.001). The strain recovered from the 6 nonclinical sealed quarters were also confirmed to be the challenge strain. In contrast, there were no surviving bacteria at the end of the experiment in the 32 remaining uninfected quarters that had been infused with seal plus lacticin 3147. Combining these results with the clinical data, 20 sealed quarters and 2 quarters treated with seal containing lacticin 3147 were either clinical or shedding the challenge strain during the experiment (Table 1). Again, this difference was significant (P<0.001). Thus, 94% of teats treated with seal plus lacticin were protected from infection with the challenge strain.

Discussion

The emergence of drug resistance among S. aureus, enterococci and pneumococci is a serious public health issue. Alternative antimicrobial treatments have been investigated by a number of groups. Mercier et al, 1997, evaluated the activities of five investigational antibiotics alone and in combinations against vancomycin- resistant Enterococcus faecium. They were, however, unable to find any combinations superior to those previously investigated. Of new and experimental drugs tested against PRP, some of the new quinolones, trovafloxacin, and oral and parenteral streptogramins are promising agents (Appelbaum, 1996). Vancomycin is still considered the best antimicrobial agent against infections caused by MRSA. However, due to the possibility of the transfer of enterococcal glycopeptide resistance to S. aureus (Noble et al, 1992), it is necessary to find alternative treatments. Combinations of vancomycin with rifampicin, fusidic acid or fosfomycin have been investigated, but the advantage of such combinations over vancomycin alone has never been demonstrated in randomised clinical trials (Michel and Gutmann, 1997). Thus, the search for alternative antimicrobials is on-going.

Bacteriocins have not previously been considered for the treatment of such infections. However, the results shown in this study suggest that lacticin 3147, which is active over a broad pH range, may prove to be a valuable addition to the limited range of therapeutic options currently available for antibiotic-resistant gram-positive infections.

The problem of antibiotic resistance can be addressed from two very different angles with the use of lacticin 3147. As discussed above, it has potential in the treatment of antibiotic resistant infections. But it may also have a role to play in reducing the occurrence of antibiotic resistance. The potential of lacticin 3147 in the prevention of mastitis has previously been demonstrated (Ryan et al, 1998). The overuse of antibiotics in veterinary medicine is thought to be a major contributing factor to the prevalence of antibiotic-resistant bacteria. Replacing antibiotics with lacticin 3147 in the treatment/prevention of mastitis would be a step towards a reduction in antibiotic use.

In vitro studies indicated that lacticin 3147 inhibits the dominant Gram-positive etiological agents of bovine mastitis namely, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalacatiae, Streptococcus bovis, and Streptococcus uberis. The efficacy of a combination of lacticin 3147 and an intramammary dry cow teat seal was assessed in animals by deliberately challenging teats of non-lactating cows with Strep, dysgalactiae. The level of challenge was deliberately set to cause approximately a 50% failure rate of the teat seal alone. Teat seal plus lacticin 3147 was significantly more successful in preventing infection (6% of quarters infected) compared to quarters containing teat seal alone (61% infected). The ability of teat seal plus lacticin 3147 to inhibit Staph. aureus was also assessed in teats of lactating cows. In one trial, the teat seal plus lacticin 3147 reduced the incidence of teats shedding Staph. aureus to 14%, compared to 66% in untreated quarters. These studies highlight the potential of lacticin 3147 as a non-antibiotic food grade anti-microbial suitable for the prevention of bovine mastitis. Lacticin 3147 also inhibits many medically important human pathogens including methicillin-resistant Staph. aureus, vancomycin-resistant Enterococcus faecalis, penicillin-resistant Pneumococcus, Streptococcus mutans and Propionibacterium acne. This work suggests that lacticin 3147 may find uses in human therapy and may provide an attractive addition to the range of anti-microbials available to inhibit antibiotic resistant Gram-positive organisms.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

References

Appelbaum, P. C. 1996. Emerging resistance to antimicrobial agents in gram-positive bacteria. Drugs 51 Suppl. 1:1-5.

Jack, R. W., J. R. Tagg, and B. Ray. 1995 Bacteriocins of gram positive bacteria. Microbiol. Rev. 59:171-200.

McAuliffe, O., M. P. Ryan, R. P. Ross, C. Hill, P. Breeuwer and T. Abee. 1998. Lacticin 3147. a broad-spectrum bacteriocin which selectively dissipates the membrane potential. Appl. Environ. Microbiol. 64:439-445.

Mercier, R-C, S. R. Pentak, and M. J. Rybak. 1997. In vitro activities of an investigational quinolone, glycycline, glycopeptide, streptogramin, and oxazolidinone tested alone and in combinations against vancomycin-resistant Enterococcus faecium. Antimicrob. Agents Chemother. 41:2573-2575.

Michel, M., and L. Gutmann. 1997. Methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci: therapeutic realities and possibilities. Lancet 349:1901-1906.

Noble, W. C, Z. Virani, and R. G. A. Cree. 1992. Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus. FEMS Microbiol. Lett. 93:195-198.

Ryan, M. P., W. J. Meaney, R. P. Ross, and C. Hill. 1998. Evaluation of lacticin 3147 and a teat seal containing the bacteriocin for the inhibition of mastitis pathogens.

Ryan, M. P., M. C. Rea, C. Hill, and R. P. Ross. 1996. An application in cheddar cheese manufacture for a strain of Lactoccus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147. Appl. Environ. Microbiol. 62:612-619. Table 1. The effectiveness of the teat seal plus lacticin 3147 formulation in eliminating Staphylococcus aureus DPC5246 in artificially infect teats of lactating cows when compared with an untreated control

Table 2. Clinical mastitis and bacterial recoveries after challenge with Streptococcus dysgalactiae in quarters treated with either seal or seal plus lacticin 3147.

Treatment Quarters Clinical infections Strep, dysgalactiae Clinical infections (no.) recoveries (d-8) and recoveries

Seal 33 16 (48.5%)' , 6 22 (66.6%)'

Seal plus lacticin 35 3 (8.6%)² 0 3 (8.6%)²

lTwo of the infections were caused by Staphylococcus aureus.

2One infection was not caused by challenge with Streptococcus dysgalactiae M. o

Claims

1. The use of lacticin 3147 in the manufacture of a medicament for the treatment prevention of infections caused by Gram-positive bacteria.

2. Use as claimed in claim 1 wherein the bacteria are multiplely drug-resistant, particularly antibiotic-resistant bacteria.

3. Use as claimed in claim 1 or 2 wherein the Gram-positive bacteria are selected from Enterococcus, Staphylococcus aureus, Pneumococcus, Propionibacterium acne, Listeria monocytogenes, Clostridium perfringens, Cloistridium dificile and Streptococcus species including Strep, dysgalactiae, Strep, agalacatiae, Strep, bovis and Strep, uberis.

4. Use as claimed in any preceding claim wherein the Gram +ve bacteria are human pathogens.

5. A pharmaceutical composition, for the treatment or prevention of infections caused by Gram-positive bacteria, comprising lacticin 3147.

6. A pharmaceutical composition as claimed in claim 5 wherein the bacteria are multiplely drug resistant, particularly antibiotic-resistant, bacteria.

9. A pharmaceutical composition as claimed in any of claims 5 to 8 further comprising a lacticin 3147 induced-bactericidal-enhancing amount of glucose.

10. A method of treatment of the human or animal body comprising application of lacticin 3147 for the prevention or treatment of bacterial infection by Gram +ve organisms.

11. A method as claimed in claim 10 further comprising the use of a lacticin 3147 induced-bacteriocidal-enhancing amount of glucose.